1. Field of the Invention
This invention relates to a filter with a definite pore size comprising fibrin and a process for the preparation thereof from fibrinogen. This invention also relates to a size selective process employing the filters of the invention.
2. Discussion of Prior Art
Formation of fibrin gels by contacting fibrinogen with a coagulation enzyme has long been known. It has been observed that when a liquid is passed over the gel, permeation of the gel increasingly becomes difficult--sometimes to the point where permeation and passage of the liquid are rapidly diminished or cease. It was believed that the gel infrastructure was extremely fragile and that the gel consisted of networks of channels and pores of varying size which were highly changeable and highly dependent upon and variable with liquid or liquid mixtures passed thereover, especially one having solid particles.
It was therefore thought that such fibrin gel was not useful in separating components where the separation was effected solely on the basis of particle size.
Specifically, when investigating the fibrin formation from fibrinogen the interest was directed to the flow properties of fibrin gels. It has e.g. been shown before that the flow properties through silica gel as well as through agar and gelatin gels are such as for a viscous flow. It has also been shown earlier that the flow through a fibrin gel is dependent upon the ionic strength and fibrinogen concentration in the preparation. In the investigations made, the permeability coefficient (K.sub.s) of the fibrin gels was determined by Poiseuille's law as follows: ##EQU1## wherein Q is the flow through the gel in cm.sup.3, A is the gel surface in cm.sup.2, .DELTA.p is the pressure difference in dynes/cm.sup.2 (=0.1N/m.sup.2 =0.1 Pa), t is the time in seconds, L is the length of the gel in cm and n is the viscosity in poise (=0.1 Pa.s.). Moreover, Kozeny-Carman has shown that the following relationship applies in a viscous or laminar flow in a capillary system: ##EQU2## wherein m is the hydraulic radius (wettable surface/wettable circumference) in cm, K.sub.o is a factor decided by the geometry of the capillaries, and .phi. is the orientation (angle) of the capillaries to the direction of flow, .epsilon. is the partial share of liquid in the gel and r is the radius of the capillaries in cm. .epsilon. can be calculated by means of the protein concentration and with a knowledge of the partial specific volume of the fibrinogen which is 0.72. For gels of the type concerned here K.sub.o and Cos .phi. cannot be calculated. In the theoretical calculations it has been assumed here that the capillaries are cylindrical and parallel to the direction of flow, which according to Madras et al brings the indicated formula to the following: ##EQU3## The theoretical pore size is therefore 2r. By effective pore size we mean: the size at which particles of smaller size pass through the pores and particles of larger size are retained. It has appeared from the tests that the clotting time (time of gel formation) of the thrombin-fibrinogen mixture, here called Ct, is directly proportional to the flow (Q) through the gel. The flow (Q) has further been found to be inversely proportional to the fibrinogen concentration (C). Provided Q=0, when 1/C=0 and Ct=0, the equation (1) will have the following form: ##EQU4## wherein k is a constant which is dependent on pH, ionic strength and calcium concentration and, moreover, is characteristic of the enzyme used in the gel formation, and Ct is the clotting time in seconds. The other symbols are the same as in equation (1). The term t is omitted when the flow is expressed in cm.sup.3 /s. According to this equation the permeability coefficient K.sub.s is thus directly proportional to the clotting time Ct and inversely proportional to the fibrinogen concentration.
By varying the pH between 6 and 10 the ionic strength between 0.05 and 0.5, the calcium ion concentration between 0 and 20 mM and/or the concentration of enzyme (e.g. thrombin, "Batroxobin" or "Arvin") between 0.01 and 10 NIH-units (or the corresponding units of other enzymes) per ml solution and the fibrinogen concentration from 0.1 and up to 40 g/l, preferably between 1 and 10 g/l, gels with K.sub.s -values [calculated according to the equation (1) ] between 10.sup.-7 and 10.sup.-12, preferably between 10.sup.-8 and 10.sup.-11, can be prepared. Calculated according to the equation (3), the corresponding average radii will be 0.03-9 .mu.m, preferably 0.09-2.8 .mu.m. If FXIII (a transamidation enzyme) and calcium ions are present in the gel formation the stability of the gels will be increased as covalent cross-linkings will arise between the chains in the subunits of the gel matrix.
Thus, now it has been found according to the invention that these fibrin gels can be used as a filter. The filter according to the invention is characterized in that it is built of fibrin and the fibrin gel is in association with a shape-retaining means which retains the shape of at least one surface of said gel against deformation when contacted by a flowing liquid.
The filter of the invention has substantially uniform pores. By that is meant that the standard deviation of pore size is less than 15 percent, preferably less than 10 percent and in some instances less than 5 percent.
The pore size of the gel has, moreover, been found to be a function of the clotting parameters used in the gels' preparation, i.e., the pore size is varied by changing said parameters. The pore size is then proportional to the clotting time.
It has now been discovered, in accordance with the invention, that fibrin in gel form can be used as a filter if means are provided to retain the shape of at least one surface of the gel against deformation when the gel is contacted by a flowing medium such as a flowing liquid medium containing components to be separated. It has also been discovered, quite surprisingly, that the gel has substantially uniform pore sizes and that these pore sizes can be regulated simply by altering the process parameters employed for the formation of the gel.
Specifically, it has been discovered if the gel is in some way stabilized by a shape-retaining means, that the gel structure is preserved and the uniform pores therein function ideally as a filter medium.
Generally speaking, the gel is brought in contact with a shape-retaining means. The shape-retaining means can be a foraminous member such as a foraminous sheet member and is preferably disposed on or in association with an upper surface of the gel, preferably in contact with the gel either directly or through an adhesive or a graft. Since the foraminous member serves to preserve the shape and structure of the upper surface of the gel when the medium to be filtered contacts the same, the gel does not collapse, thereby allowing the uniform pores thereof to function ideally as a filter medium.
Foraminous members functioning as a shape-retaining means can have virtually any size and shape, although they are preferably in the form of a sheet and preferably are substantially co-extensive with the upper surface of the gel. The foraminous sheet members can be in the form of a fibrous network such as in the form of a woven or non-woven or knitted fabric, the fibers of which can be natural or synthetic.
When the fibers of a foraminous sheet member are natural, they can be, for example, made of silk, wool, cotton, cellulose, hemp, jute or the like.
As synthetic fibers, there are contemplated in particular fibers made of nylon, polyester, polyolefin, fibers made of vinyl polymers, acrylics such as polyacrylonitrile, rayon, to name a few.
The fibers generally have a thickness between 1 .mu.m and 1000 .mu.m, preferably between 10 and 20 .mu.m, and are disposed in relationship to one another to define openings therebetween of between 0.01 and 5 mm, preferably between 0.05 and 1 mm, it being understood that the size of the openings between the fibers of the foraminous sheet is not especially critical, provided it allows passage therethrough of the medium to be filtered. It is preferred that as much fiber be in contact with or adhere to the gel as possible so as to insure maximum structural integrity of the surface of the gel initially to come in contact with the medium to be filtered.
Instead of using a fibrous foraminous member, one can use one made of wires, such as wires made of copper, tin, zinc, aluminum, glass, boron, titanium, steel, stainless steel, etc. The wires function analogously to the function performed by the fibers in providing structural integrity to at least one surface of the gel, preferably the upper surface or surface which is to be initially brought in contact with a mixture to be filtered. The interstices between the wires are of the same magnitude as the interstices between the fibers of a woven, non-woven or knitted fabric serving as a foraminous sheet member. The wires can be in the form of a screen, wire mesh or an expanded wire sheet and are preferably co-extensive with at least one side of the gel, preferably the upper surface.
The gel has uniform pores but owing to the manner by which the gel can be formed, can have uniform pores over a wide range. Preferably, the substantially uniform pores of the fibrin gel have a theoretical pore size or diameter in the range of about 0.003 to 1 .mu.m, more preferably 0.009 to 0.3 .mu.m.
The gel is formed by contacting fibrinogen with an enzyme, especially a coagulation enzyme. Particularly contemplated enzymes for use in forming a fibrin gel include thrombin, Batroxobin, Arvin, Eccarin, Staphylocoagulase, Papain, Trypsin, caterpillar venom enzyme, etc.
Generally speaking, the gel formation is effected at room temperature, although temperatures from -3.degree. C. up to 58.degree. C. can be employed. Preferably, the temperature is in the range of 0.degree. to 40.degree. C.
It is preferred that the gel is formed by contacting the fibrinogen with an enzyme in the presence of calcium ions. The calcium ion concentration can be up to 20 mM. The presence of calcium ions is not required in all instances. Where thrombin is employed as the coagulation enzyme, the gel can be formed in the absence of a calcium ion.
In forming the gel, there is generally employed 0.1 to 10.sup.-5 enzyme units per gram fibrinogen, preferably 10 to 10.sup.-3 enzyme units per unit weight fibrinogen. Following formation of the gel whose coagulation time is a function of the relative amount of enzyme to fibrinogen as well as the concentration of calcium ion, the gel is preferably hardened or set by crosslinking the components thereof by contacting the gel with a crosslinking agent. Crosslinking agents contemplated include bis-imidates such as suberimidate, azides like tartryl di(.epsilon.-amino carproylazide), aryl dihalides like 4,4-difluoro-3,3'-dinitrophenyl sulfone, glutardialdehyde, nitrenes, N,N'(4-azido-2-nitrophenyl)-cystamine dioxide, cupric di(1,10-phenanthroline), dithio bis-(succininimidyul propionate), N,N'-phenylene dimaleimide as well as polyethyleneimides and other bifunctional compounds, especially those known to crosslink with epsilon lysine, alpha amino groups, carboxy groups of aspartic and glutamic acids, and hydroxyl groups of amino acids in the protein chain (e.g. threonine and serine).
Bis-imidates which can be used include those of the formula ##STR1## wherein n=3 to 15 especially 3 to 10.
Azides which can be used include substituted and unsubstituted azides of the formula ##STR2## wherein n-1 to 20 especially 1 to 15. Azides contemplated include those having a hetero atom in the chain, especially nitrogen. Also contemplated are hydroxy substituted azides.
Aryl dihalides which can be used include those having mono, poly and fused rings as well as rings joined by a direct bond or through a methylene bridge or a sulfo bridge. The halogen of the halide can be fluorine, chlorine, or bromine. The compounds can be substituted by inert or functional groups such as nitro, or disulfide. Contemplated compounds include those where a functional group has replaced one of the halo substituents, e.g. nitrio. Compounds contemplated include ##STR3## Especially contemplated is glutardialdehyde.
Generally speaking, the crosslinking agent is employed in an amount of between 0.001% and 8% by weight, preferably between 1 and 2% by weight of the gel for 1-120 minutes. Crosslinking is effected at temperatures of between 10.degree. and 40.degree. C., preferably 20.degree. to 25.degree. C. After the hardened or crosslinked structure is obtained, the gel is usually washed free of extraneous material.
The gel in such hardened form is useful as a filter, i.e., without any foraminous sheet material. Preferably, however, the gel is formed on or in association with a shape-retaining means such as a net, wire mesh or other sheet material and while in contact with such shape-retaining means is hardened by the use of a hardening or crosslinking agent.
Preferably, the gel is supported on its upper and lower surfaces by a shape-retaining means such as a foraminous sheet or the like, whereby to insure that the gel retains its shape during use as a filter.
This invention further contemplates a process for separating a first substance having a theoretical size of 0.003 to 1 .mu.m from a second substance having a larger size which comprises passing a mixture of said first and second substances over a filter comprising fibrin in gel form and having pores of substantially uniform size, said filter having means for retaining the shape of at least one surface of said gel against deformation when contacted by a flowing medium, wherein the effective pore size of said fibrin gel is larger than the particle size of said first substance and smaller than the particle size of said second substance. Preferably, the pores of the gel have a theoretical size of 0.009 to 0.3 mm.
The filters of the invention are important, as they permit the separation of bacteria and viruses from mixtures containing the same. The ability to regulate the pore size and to achieve a gel of uniform pore size is an important and critical characteristic of the filters of the invention. These filters permit the separation of blood components, the separation of components of blood plasma, the removal of platelets from blood, the fractionation of cells and cell fragments and the separation of high molecular weight protein aggregates. In addition, a variety of particles such as latex, silica, carbon and metallic particles may be separated over these filters. Components which can be separated include those shown in the table below:
TABLE A __________________________________________________________________________ Effective Material from Pore Size Material A Which Material How Separated Range for Separated "A" is Separated Retained Eluted Filter __________________________________________________________________________ Blood platelets Blood plasma X Below 1 .mu.m Red blood cells " X 1 .mu.m and below Sendai virus Culture medium X 0.1 .mu.m and below Sendai virus " X 0.2 .mu.m and above Liver mito- Cyto plasma X 0.5 .mu.m and chondria below Liver mito- " X 0.5 .mu.m and chondria above Adeno virus Culture medium X 0.05 .mu.m and below Adeno virus " X 0.1 .mu.m and above E. coli bacteria " X 1 .mu.m and below FVIII complex High molecular X X 0.05 .mu.m and weight material (h.m.w) (l.m.w) below (h.m.w) separ- ated from low mo- lecular weight ma- terial (l.m.w) Blood leucocytes Blood plasma X 1 .mu.m and below Blood lymphocytes " X 1 .mu.m and below __________________________________________________________________________
Fibrin gel filters have above all the advantages over other gel filters that the pore size can be simply varied as desired. Moreover, the present filters have high flow rates at such pore sizes as can be used to remove very small particles, such as virus particles. In this respect, the filters of the invention are more suitable than known membrane filters and filters of polyacrylamide gels. The absence of absorption of protein on the filters is also an advantage as compared with certain other filters.